The long term goal of our research is to understand how a complex nervous system with millions of specialized synaptic connections is properly "wired" during development. We want to understand the molecular mechanisms of recognition, adhesion, repulsion, and signaling that confer the incredible specificity that is observed during this process of growth cone guidance. Since many neurological diseases, mental health disorders, and birth defects may originate from improper wiring of the nervous system, it is clearly advantageous for us to gain a full understanding of the molecular mechanisms responsible for generating this complex pattern of neuronal connections. Our approach is to study this process in Drosophila, where a powerful combination of genetic, molecular, and cellular approaches can be employed. We have focused on the behavior of growth cones at the midline of the Drosophila embryonic CNS, an excellent model system with significant advantages for a detailed dissection of growth cone guidance mechanisms. We want to understand how commissural neurons are attracted towards and ultimately cross the midline of the CNS, and conversely, we want to understand how ipsilaterally projecting neurons are kept on their own side and prevented from crossing the midline. Since this fundamental behavior of growth cones at the CNS midline is conserved from Drosophila to humans, our work in Drosophila has broad applications. This proposal is focused on the commissureless (comm) gene, the most promising entry point in our dissection of commissural growth cone guidance. In comm mutant embryos the CNS lacks most if not all commissural axon pathways. Neurons, such as RP1, that normally project their axons across the midline fail to do so in these mutant embryos; instead they make an ipsilateral projection that is normal except for the failure to cross the midline. This and other observations have led us to hypothesize that this mutation disrupts a critical component of the commissural growth cone guidance mechanism, most likely a signal, receptor, or a component of a signal transduction pathway. We propose to test this hypothesis through the molecular cloning and characterization of the comm gene. In addition, we propose to identify other components of this pathway by using genetic screens to isolate enhancers and suppressors of the comm phenotype and by expression cloning/biochemical approaches to isolate proteins that interact directly with the Comm protein. Our specific aims are to 1) identify and clone the DNA sequences encoding the comm transcription unit; 2) determine the nature of the comm gene product by sequencing comm cDNA clones; 3) generate antibodies against the comm protein to determine its spatial pattern of expression/accumulation in the embryonic CNS; 4) determine the sites of comm function by mosaic analysis; 5) determine the epistatic relationship of comm to other genes involved in commissural growth cone guidance; 6) isolate genetic enhancers and suppressors of the comm CNS phenotype; and 7) identify other proteins that interact with Comm protein using appropriate expression cloning/biochemical approaches.